T-PCR Method To Detect ASFV, SVDV, FMDV-O, PRV’s Preliminary Study

ASFV, SVDV, FMDV-O, PRV are four important swine pathogen. Although there is no reports of ASF occur current,due to Russia’s neighbors repeatedly reported occurrence of the disease, and its possible serious consequences,it is necessary to establish a detection method for the prevention of the disease in advance. And it can provide a test basis for future research of the disease. On clinical manifestations,SVD is very similar to FMD. It will be very difficult to distinguish between them by clinical symptoms. So it is significant for treatment and prognosis for the future, if we could establish a rapid and accurate method for the detection and identification of these two diseases.In addition, clinical investigations in this laboratory demonstrate that PR has been high incidence of herds in recent years. The adverse clinical manifestations come bring huge economic losses to livestock breeding industry.In this study, four target gene fragment was cloned by using pMD19-T,and got four positive recombinant plasmids:T/ASFV, T/FMDV-O, T/PRV, T/SVDV.Based on the analysis ASFV, SVDV, FMDV-O, PRV virulence factor genes and the principle of target enrichment multiplex PCR (Tem-PCR),we design four pairs of specific nested PCR primers and probes, and adding a tag sequence that can be recognized by universal primers at the 5’end of each of the inner primer. By adjusting and optimizing the impact of amplification of nested PCR primer mixture concentration, annealing temperature and reaction parameters, and the amplification products were detected by microarray, establishment Tem-PCR method of detecting four kinds of common swine pathogen simultaneously eventually.After optimization, the optimum concentration Primer Mix is 0.2μM and the annealing temperature of the reaction was 50 ℃ of the Tem-PCR method. The results show that:ASFV, FMDV-O, PRV can be detected within a reaction tube quickly and simultaneously by using this method,and obtain specific product,the size were 272bp(ASFV),576bp(PRV) 233bp(FMDN-O). The sensitivity were:3.5 ×106 copies/μL (ASFV),4.2×106 copies/μL (FMDV-O),4.6×103copies/μL (PRV). Specificity of the test found that the Tem-PCR method from the positive recombinant plasmid T/ASFV, T/FMDV-O, T/PRV genome have been a lot of specific amplification products, other genomic DNA without specific bands appear.Using the optimized Tem-PCR method amplify T/ASFV, T/FMDV-O, T/PRV, T/SVDV, and the product was detected by gene chip technology, the results show that all the hybrid quality control sites and samples were issued green fluorescence, all negative control sites were not fluoresce, indicating hybridization process is effective and reliable.